The pathogenesis of alcoholic liver disease (ALD) is multi-factorial. Animal and human studies suggest that gut-derived endotoxin is a critical factor contributing to ALD. Endotoxins are derived from the cell wall of Gram-negative bacteria, and normally penetrate the gut epithelium in only trace amounts due to tightly regulated intestinal barrier function. However, endotoxin leakiness is increased under pathological conditions, such as alcohol abuse. The barrier function of the intestinal epithelium is provided by paracellular apical junction complexes, including tight junctions and adherens junctions located at the apical end of epithelial cells, and by mucus layers. Protection of the mucus layer involves mucins and a number of regulatory peptides, such as intestinal trefoil factors (ITFs), which play critical rols in formation and stabilization of the barrier, enhancement of rapid intestinal repair ('restitution), modulation of mucosal differentiation processes, and modulation of the mucosal immune response. Formation and restitution of the barrier is also dependent on uninterrupted mucosal blood flow and ongoing glycolysis. Intestinal epithelial cells are positioned between an anaerobic lumen and a highly metabolic lamina propria forming a steep physiologic oxygen gradient. Physiological and pathological hypoxia induces hypoxia-inducible factor (HIF) -1 and -2, which represent a major adaptation response by upregulating a variety of gene products such as intestinal ITF, mucins and glycolytic enzymes. Our preliminary studies showed that chronic alcohol feeding decreased intestinal HIF-2, ITF and tight junction expression leading to increased endotoxemia and ultimately liver injury. Supplementation of a probiotic strain, lactobacillus rhamnosus GG (LGG), attenuates those deleterious effects and ALD. However, the mechanisms are unknown. Our central hypothesis is that alcohol feeding causes a change favoring oxidative stress, inflammation and mitochondrial damage, which reduces HIF responsive signaling and the targeted proteins, leading to disruption of gut barrier function, increased endotoxemia and subsequent ALD, and that probiotic supplementation will attenuate alcohol-induced liver injury by potentiating HIF regulated responses. To test this hypothesis, we will carry our following specific aims: Specific Aim 1. Determine the effects of ethanol and probiotics on intestinal HIF signaling and mechanisms in ALD. Specific Aim 2. Determine whether HIF signaling is required for the protective effect(s) of probiotic supplementation against alcohol-induced liver injury. We will use intestine-specific HIF-1 and HIF-2 knockout mice to evaluate and compare the role of HIF-1 and HIF-2 in ALD. We will then use prolyl-4 hydroxylase-1 PH.D.1 knockout mice for a gain-of-function strategy to determine whether an increase HIF attenuates alcohol-induced mucus layer dysfunction in ALD. This study will have a major impact on the development of new therapeutic targets for the prevention and treatment of ALD.